Matthew J. Cornish and Drs. Matthew L. Madsen and Daniel L. Simmons, Zoology
Organisms are equipped with an amazing ability to deal with situations which its environment may present. There are biochemical pathways responsible for monitoring and responding to environmental changes. Two examples are protein induction and inhibition. In the last few years, however, signaling through pre-mRNA splicing has been extensively explored.
Splicing refers to the process of shortening pre-mRNA to functional mRNA by removing introns. The spliced MRNA can then be translated into active proteins. Splicing of premRNA determines the conformation of proteins. Dr. Simmons identified the protein prostaglandin G/H synthase 2, also known as cyclooxygenase 2 or COX-2.1 COX-2 is induced by factors that cause cells to divide. The COX-2 gene in chicken is unique in that its first intron is retained longer than the other introns and it does not utilize the traditional AG-dependent splicing machinery. By characterizing this chicken COX-2 intron 1 splicing mechanism, we are accomplishing two feats. First, we will broaden the understanding of biochemical signals and their relationship with the environment. Second, we will specifically define one of the physiological controls used by chickens during cell growth and cancer defense.
In order to characterize this splicing mechanism, we first had to develop a system that would allow us to detect and measure splicing in vivo. To do this, we have manipulated a gene called luciferase. Luciferase is the gene which allows fireflies to glow. The amount of light emitted by luciferase activity can be measured. Using this gene, we have engineered a plasmid, or DNA strand, which is a fusion of several genes. The sequence includes a promoter region used by cells to recognize transcription domains. We inserted the first part of the chicken COX-2 gene including intron 1 into the plasmid. Following the COX-2 gene fragment is the luciferase gene. In order to get luciferase activity, the luciferase portion of the clone must be in-frame. In our construct, however, only COX-2 is in frame while the downstream luciferase gene is shifted out-of-frame. But, when intron I is spliced out of COX2, luciferase shifts into frame and is translated as a functional protein. By measuring the light emitted, we can quantitatively show splicing activity. To date, we have used this construct in a number of ways.
The first experiment done with this plasmid was to measure splicing activity in chicken embryo fibroblasts (CEF). Transformation of CEF with the Rous sarcoma virus causes the cells to become cancerous. Splicing activity was measured in transformed and nontransformed cells. The data show that the COX-2 intron 1 is spliced out in the transformed cells. This is exciting because the data suggest that intron I is providing another specific control over cell division.
Next, we transfected the same clone into other non-chicken cell lines. The data clearly indicate that splicing of this chicken clone actually occurs in other species. However, no other species is known to have an intron that uses this same splicing mechanism. Note, though, that the splicing is not nearly as efficient as it is in chicken. Two hypotheses to explain decreased splicing activity include variations in splicing machinery between species and a lack of recognition of the chicken enhancer sequence.
Recent data, suggest the presence of an enhancer region in the RNA downstream from intron 1. We showed this by making a new construct which is the same as the one in Table 1, A and B without the last 280 bases of COX-2. When this construct was transfected into CEF, the splicing efficiency dropped a surprising 50-fold. The putative enhancer region is located somewhere within the last 280 bases of our COX-2 gene fragment.
In summary, my research showed that this newform of splicing in pre-mRNA is present in all organisms examined. The implication of this important finding is that this splicing mechanism has played an important role in evolution. In addition, the enhancer region likely plays a role in all higher lifeforms. The next step in this project is to specifically isolate the bases which control splicing, including the enhancer region. This will require mutating our original construct, and then cloning, sequencing, transfecting, and assaying the mutants for luciferase activity. In addition, we will search computer databases in order to identify other genes that may use this same form of splicing.2
References
- Xie, W. et. al., Proc. Natl. Acad. Sci. USA 88, (1991) 2692-2696.
- I am particularly grateful to Dr. Simmons for his advice, Matt Madsen for his assistance, and my wife Shanine for her patience, love,
and support.